HIGH-EFFICIENCY OF GLYCEROL 2-PHOSPHATE AND SN-GLYCEROL 3-PHOSPHATE AS NUCLEOTIDYL ACCEPTORS IN SNAKE-VENOM PHOSPHODIESTERASE ESTERIFICATIONS - FORMATION OF PRIMARY AND SECONDARY AMP-O-GLYCERYL AND AMP-O-GLYCEROPHOSPHORYL ESTERS AND EVIDENCE FOR AN ACCEPTOR-BINDING ENZYME SITE

Snake venom phosphodiesterase (SVP) catalyzes the alcoholysis of ATP b y primary R-CH2OH alcohols with uncharged R residues, yielding AMP-O-C H(2)R esterification products. The alcohols compete with water for an SVP-bound adenylyl intermediate. In this study, it has been shown that SVP also catalyzes the reactions of glycerol 2-phosphate and sn-glyce rol 3-phosphate with ATP to yield AMP-O-glycerophosphoryl esters. The products were identified by HPLC, the dependency of the reactions on g lycerol phosphates, ultraviolet spectroscopy, and conversion to AMP by phosphodiesterase, or to AMP-O-glyceryl esters by alkaline phosphatas e. The results demonstrated that R-CH2OH alcohols with negatively char ged R residues, as well as secondary alcohols, act as adenylyl accepte rs in SVP reactions: thus extending the usefulness of SVP as a tool to produce 5'-nucleotide derivatives. The efficiencies (E(A)) of glycero l phosphates as adenylyl accepters were very high at low, millimolar c oncentrations, but decreased abruptly when the acceptor concentration was increased and, for glycerol 2-phosphate, when P-i or NaCl was pres ent. In contrast, glycerol E(A) was independent of its own concentrati on, P-i, and NaCl. The responses of glycerol phosphates indicate that they act as adenylyl accepters via a mechanism different from uncharge d R-CH2OH alcohols. The occurrence of an acceptor-binding enzyme site, specific for negatively charged R residues, and its potential relevan ce to the in vivo role of 5'-nucleotide phosphodiesterases as 5'-nucle otidyl transferases are discussed.